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Synthetic method for L-carnitine

A technology of L-carnitine and synthesis method, which is applied in the field of medicine, can solve the problems of high solvent use and production cost, lack of market competitiveness of products, low conversion rate and yield, etc., to improve market competitiveness, reduce production steps, The effect of mild reaction conditions

Active Publication Date: 2014-04-09
ENZYMEWORKS
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] (3) Biosynthesis of different starting materials, such as enzymatic resolution of DL-carnitine, enzymatic conversion of β-dehydrocarnitine, and enzymes of trans-crotonobetaine and γ-butylbetaine Preparation by method, etc., due to high production cost, it is difficult to be widely used; and fermentation method using ethyl 4-chloroacetoacetate as raw material, but the conversion rate and yield are low (Chinese patent 200810121542.9)
For example, Chinese patent 201310132479.x uses ketoreductase expressed by recombinant Escherichia coli to prepare (R)-4-chloro-3-hydroxy-butyric acid ethyl ester; but it has the following problems: 1. The substrate concentration is low; 2. , high enzyme dosage; 3, it can only generate the key intermediate of L-carnitine, and after extraction, subsequent reactions can be used to obtain L-carnitine, the use of solvents and production costs are high, and the product lacks market competitiveness

Method used

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Experimental program
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Embodiment 1

[0028] The preparation of embodiment 1 ketoreductase

[0029] Inoculate a single colony of recombinant Escherichia coli containing the ketoreductase gene from a glycerol tube or transformation plate into 4 mL of liquid LB medium containing ampicillin resistance and activate overnight (35°C, 220rpm). Transfer 50 mL of liquid LB medium containing ampicillin resistance from the overnight culture at 1 / 100 inoculum, culture at 35°C and 220 rpm until OD 600 When the value reaches 0.6-0.8, add IPTG and continue culturing overnight at 30°C. Cells were collected by centrifugation, and suspended in 10 mL of phosphate buffer (2 mM, pH 7.0). The cell suspension was ultrasonically broken in an ice bath for 15 minutes, centrifuged, the supernatant was pre-frozen overnight, and freeze-dried for 24-40 hours to obtain ketoreductase in the form of freeze-dried powder.

Embodiment 2

[0031] 1) Add ethyl 4-chloroacetoacetate (1.5g, 9.11mmol) into a 50mL round bottom flask, add 20mL phosphate buffer (0.1M, pH6.5), then add 3mL isopropanol and 45mg ketone in sequence Reductase powder and 4.5mg NAD + The dry powder was stirred magnetically at 30°C to start the reaction; at the same time, the pH was maintained at 6.5 using a titrator. GC monitoring, after 30 hours of reaction, the conversion rate was greater than 99%, and the reaction was terminated for use.

[0032] 2) Sodium hydroxide (0.73g, 18.2mmol) and trimethylamine aqueous solution (1.44mL, 10.9mmol) with a mass ratio of 25% were prepared as a mixed solution, and slowly added dropwise to the above-mentioned enzymatic reaction at 0-5°C In the aqueous solution, the addition was completed in about 3 hours, and the stirring reaction was continued at 0-5°C for 24 hours. After the conversion rate was detected by HPLC >99%, the reaction stopped. Add about 3.68mL of hydrochloric acid (concentration: 36%) to n...

Embodiment 3

[0034] 1) Add ethyl 4-chloroacetoacetate (5g, 30.35mmol) to a 100mL round bottom flask, add 40mL phosphate buffer (0.1M, pH6.5), then add 5mL isopropanol and 100mg ketone to reduce Enzyme powder and 10mg NAD + Dry powder, stir at 30°C to start the reaction; at the same time, use a titrator to maintain the pH at 6.5. GC monitoring, after 48 hours of reaction, GC detected that the conversion rate was greater than 99%, and the reaction was terminated for use.

[0035] 2) Sodium hydroxide (2.43g, 60.7mmol) and trimethylamine aqueous solution (21.3mL, 36.12mmol) with a mass ratio of 25% were prepared as a mixed solution, and slowly added dropwise at 0-5°C to the above enzyme-reacted In the aqueous solution, the addition was completed in about 3 hours, and the stirring reaction was continued at 0-5°C for 24 hours. After the conversion rate was detected by HPLC >99%, the reaction stopped. About 12.3 mL of hydrochloric acid (concentration: 36%) was added to neutralize to pH = 6. Aft...

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Abstract

The invention relates to a synthetic method for L-carnitine. L-carnitine is obtained by the steps of dissolving a substrate 4-chloracetyl acetidin in a phosphate buffer at a temperature of 25-35 DEG C; reacting the substrate for 20-50 hours at a pH of 6-7 under the effects of a ketoreductase, a co-enzyme and isopropanol to form (R)-4-chloro-3-hydroxyl-ethyl butyrate; dissolving sodium hydroxide in a trimethylamine aqueous solution with a mass ration of 25% to form a mixed solution; then dropwise adding the mixed solution in (R)-4-chloro-3-hydroxyl-ethyl butyrate slowly at a temperature ranging from -5 DEG C to 5 DEG C; reacting for 20-30 hours at the temperature ranging from -5 DEG C to 5 DEG C; stopping the reaction; adjusting a pH value to 5-7; and the purifying by centrifugation, ultra filtration and resin purification. The synthetic method improves a production flow, increases substrate concentration, reduces dosage of enzyme, reduces production cost and increases market competitiveness of products.

Description

technical field [0001] The invention belongs to the technical field of medicine, and in particular relates to a synthesis method of L-carnitine. Background technique [0002] L-carnitine, also known as L-carnitine, the trade name is also called Weikangsu, vitamin Bt, the English name is L-Carnitine, the chemical name is: (R)-3-hydroxy-4-trimethylammonium butyric acid inner salt or β-Hydroxy-γ-trimethylammonium butyric acid inner salt, molecular formula C 7 h 15 NO 3 , molecular weight: 161.20. [0003] L-carnitine is a special amino acid that widely exists in human body tissues and is an essential nutrient for humans and animals. Its basic function is to assist long-chain fatty acids to penetrate the inner mitochondrial membrane for oxidation. It is clinically used for carnitine deficiency and carnitine deficiency in long-term renal dialysis patients. It can also be widely used in the treatment of various ischemic heart diseases. It is good for shock, acute and chronic ...

Claims

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Application Information

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IPC IPC(8): C07C229/22C07C227/08C12P7/62
Inventor 陶军华李斌邓阳生唐苏苏鞠鑫
Owner ENZYMEWORKS
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